Understanding the interactions between cells, extracellular matrix, and stromal molecules in a tissue environment is an emerging frontier of biology. While such interactions are new, providing images of biological tissues is a time-honored endeavor in the field of biology. An optical microscope, i.e., a type of microscope which uses visible light and a series of lenses to magnify images of small objects, cannot provide sufficient information needed by researchers and clinicians about a host of biological tissues. Accordingly, special nonlinear optical microscopic techniques have been developed for different modalities for imaging complex tissue samples with inherent 3D spatial resolution requirements. Normally laser beams are used to excite tissue samples in different modalities. Some modes of nonlinear optical microscopy involve a single laser beam for excitation of the biological tissue. While other modes involve multiple beams. Single beam modalities include Two-Photon Fluorescence (TPF) microscopy, Second Harmonic Generation (SHG), and Third Harmonic Generation (THG) microscopy. TPF and SHG can be integrated with a single femtosecond (fs) laser. THG microscopy has also been combined with SHG and TPF by using an Optical Parametric Oscillator (OPO) system.
Coherent anti-Stokes Raman Scattering (CARS) microscopy is another nonlinear optical imaging technique that facilitates high-speed vibrational imaging of molecules. As a two-beam modality, CARS microscopy is mostly operated with picosecond (ps) pulses, either from two synchronized Ti:sapphire lasers or from a synchronously pumped OPO system. In comparison with fs pulses, ps pulse excitation not only provides sufficient spectral resolution, but also increases the ratio of resonant signal to nonresonant background.
Recently, CARS, TPF, and sum-frequency generation (SFG) modalities have been integrated into a microscope operated with ps pulses for multimodal imaging of white matter and arterial tissue. Although tunable ps laser systems operating in the near infrared (NIR) range are widely accepted for high-speed CARS imaging, the reduced efficiency of non-linear optical (NLO) process caused by longer pulse duration hinders the application of ps lasers to TPF and SHG imaging. While, a key advantage of the fs laser source is its superior image capabilities of TPF, SHG, and THG imaging over ps lasers, CARS microscopy has traditionally been performed with ps laser systems.
Therefore, a practical and efficient solution to fully utilizing all NLO imaging capabilities is therefore needed.